Human short-chain dehydrogenase

ABSTRACT

The invention provides a human short-chain dehydrogenase (HSCD) and polynucleotides which identify and encode HSCD. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for treating or preventing disorders associated with expression of HSCD.

This application is a divisional application of U.S. application Ser.No. 09/019,216, now U.S. Pat. No. 5,928,923 filed Feb. 5, 1998.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of ahuman short-chain dehydrogenase and to the use of these sequences in thediagnosis, treatment, and prevention of immune disorders and cancer.

BACKGROUND OF THE INVENTION

Acetyl CoA is a key intermediate in the mitochondrial metabolism ofpyruvate and fatty acids. Pyruvate which is generated in the cytosolduring glycolysis, is transported across the mitochondrial membranes tothe interior mitochondrial matrix. The complete oxidation of pyruvate toform CO₂ and H₂O occurs in the mitochondrion and utilizes O₂ as thefinal electron acceptor (oxidizer). Immediately on entering the matrix,pyruvate reacts with coenzyme A to form CO₂ and the intermediate acetylCoA, a reaction catalyzed by the enzyme pyruvate dehydrogenase. Thisreaction is highly exergonic (ΔG°=−8.0 kcal/mol) and is essentiallyirreversible. Pyruvate dehydrogenase is one of the most complex enzymesknown. The molecule is 30 nm in diameter and contains 60 subunitscomposed of three different enzymes, several regulatory polypeptides,and five different coenzymes. Fatty acids are also oxidized in themitochondrion to produce acetyl CoA; the energy released is used tosynthesize ATP form ADP and phosphate ion. In eucaryotic cells, fattyacids containing approximately 20 CH2 groups are degraded chiefly inperoxisomes and converted to acetyl CoA.

Fatty acids are stored as triglycerols, primarily as droplets in adiposecells. In response to hormones such as adrenaline, triglycerols arehydrolyzed in the cytosol to free fatty acids and glycerol. Fatty acidsare released into the blood, where they are taken up and used by mostcells. They are the major energy source for many tissues, in particular,heart muscle. In humans, the oxidation of fats is quantitatively moreimportant than the oxidation of glucose as a source of ATP, due to thefact that oxidation of 1 gram of triacylglycerol to CO₂ generates aboutsix times as much ATP as does the oxidation of 1 gram of hydratedglycogen.

Nicotinamide adenine dinucleotides are involved in a very large numberof oxidoreduction reactions both in the cytosol and in mitochondria,including the oxidation of the acetyl group of acetyl CoA to CO₂. Ingeneral, nicotinamide adenine dinucleotides are not tightly bound toenzymes and may function as substrates, although they are often referredto as coenzymes. Nicotinamide adenine dinucleotide (NAD⁺) andnicotinamide adenine dinucleotide phosphate (NADP⁺) undergo reversiblereduction to NADH and NADPH, respectively, but have different activitiesin the cell. The major role of NADH is to transfer electrons frommetabolic intermediates in a large number of biosynthetic processes intothe electron transfer chain. NADPH acts as a reducing agent in a largenumber of biosynthetic processes.

In the cytosol, free fatty acids are linked to coenzyme A to form anacyl CoA in an energetic reaction coupled to the hydrolysis of ATP toAMP and inorganic pyrophosphate. The fatty acyl group is thentransported across the inner mitochondrial membrane by a transporterprotein and is reattached to another CoA molecule on the matrix side.Each molecule of acyl CoA in the mitochondrion is oxidized to form onemolecule of acetyl CoA and an acyl CoA shortened by two carbon atoms.Concomitantly, NAD⁺ and FAD are reduced to NADH and FADH₂, respectively.This set of reactions is repeated on the shortened acyl CoA until allcarbon atoms are converted to acetyl CoA. Short-chain acyl-CoAdehydrogenase (SCAD) is a homotetrameric mitochondrial flavoenzyme thatcatalyzes the initial reaction in short-chain fatty acid beta-oxidation.Defects in the SCAD enzyme are associated with neuromusculardysfunction. (See, e.g., Corydon, M. J. et al. (1997) Mamm Genome8(12):922-926.)

The discovery of a new human short-chain dehydrogenase and thepolynucleotides encoding it satisfies a need in the art by providing newcompositions which are useful in the diagnosis, treatment, andprevention of immune disorders and cancer.

SUMMARY OF THE INVENTION

The invention features a substantially purified polypeptide, humanshort-chain dehydrogenase (HCSD), comprising the amino acid sequence ofSEQ ID NO:1 or a fragment of SEQ ID NO:1.

The invention further provides a substantially purified variant havingat least 90% amino acid sequence identity to the amino acid sequence ofSEQ ID NO:1 or a fragment of SEQ ID NO:1. The invention also provides anisolated and purified polynucleotide encoding the polypeptide comprisingthe sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The inventionalso includes an isolated and purified polynucleotide variant having atleast 90% polynucleotide sequence identity to the polynucleotideencoding the polypeptide comprising the amino acid sequence of SEQ IDNO:1 or a fragment of SEQ ID NO:1.

Additionally, the invention provides a composition comprising apolynucleotide encoding the polypeptide comprising the amino acidsequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. The inventionfurther provides an isolated and purified polynucleotide whichhybridizes under stringent conditions to the polynucleotide encoding thepolypeptide comprising the amino acid sequence of SEQ ID NO:1 or afragment of SEQ ID NO:1, as well as an isolated and purifiedpolynucleotide which is complementary to the polynucleotide encoding thepolypeptide comprising the amino acid sequence of SEQ ID NO:1 or afragment of SEQ ID NO:1.

The invention also provides an isolated and purified polynucleotidecomprising the polynucleotide sequence of SEQ ID NO:2 or a fragment ofSEQ ID NO:2, and an isolated and purified polynucleotide variant havingat least 90% polynucleotide sequence identity to the polynucleotidecomprising the polynucleotide sequence of SEQ ID NO:2 or a fragment ofSEQ ID NO:2. The invention also provides an isolated and purifiedpolynucleotide having a sequence complementary to the polynucleotidecomprising the polynucleotide sequence of SEQ ID NO:2 or a fragment ofSEQ ID NO:2.

The invention further provides an expression vector containing at leasta fragment of the polynucleotide encoding the polypeptide comprising thesequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1. In another aspect,the expression vector is contained within a host cell.

The invention also provides a method for producing a polypeptidecomprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQID NO:1, the method comprising the steps of: (a) culturing the host cellcontaining an expression vector containing at least a fragment of apolynucleotide encoding the polypeptide comprising the amino acidsequence of SEQ ID NO:1 or a fragment of SEQ ID NO: 1 under conditionssuitable for the expression of the polypeptide; and (b) recovering thepolypeptide from the host cell culture.

The invention also provides a pharmaceutical composition comprising asubstantially purified polypeptide having the sequence of SEQ ID NO:1 ora fragment of SEQ ID NO:1 in conjunction with a suitable pharmaceuticalcarrier.

The invention further includes a purified antibody which binds to apolypeptide comprising the sequence of SEQ ID NO:1 or a fragment of SEQID NO:1, as well as a purified agonist and a purified antagonist of thepolypeptide.

The invention also provides a method for treating or preventing acancer, the method comprising administering to a subject in need of suchtreatment an effective amount of an antagonist of the polypeptide havingthe amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1.

The invention also provides a method for treating or preventing animmune disorder, the method comprising administering to a subject inneed of such treatment an effective amount of a pharmaceuticalcomposition comprising substantially purified polypeptide having theamino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1, anantagonist of the polypeptide having the amino acid sequence of SEQ IDNO:1 or a fragment of SEQ ID NO:1.

The invention also provides a method for detecting a polynucleotideencoding a polypeptide comprising the amino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO: 1 in a biological sample containingnucleic acids, the method comprising the steps of: (a) hybridizing thecomplement of the polynucleotide encoding the polypeptide comprising theamino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 to atleast one of the nucleic acids of the biological sample, thereby forminga hybridization complex; and (b) detecting the hybridization complex,wherein the presence of the hybridization complex correlates with thepresence of a polynucleotide encoding the polypeptide comprising theamino acid sequence of SEQ ID NO:1 or a fragment of SEQ ID NO:1 in thebiological sample. In one aspect, the nucleic acids of the biologicalsample are amplified by the polymerase chain reaction prior to thehybridizing step.

BRIEF DESCRIPTION OF THE FIGURES

FIGS. 1A, 1B, 1C, and 1D show the amino acid sequence (SEQ ID NO:1) andnucleic acid sequence (SEQ ID NO:2) of HSCD. The alignment was producedusing MACDNASIS PRO™ software (Hitachi Software Engineering Co. Ltd.,San Bruno, Cailf.).

FIG. 2 shows the amino acid sequence alignments among HSCD (35635 1; SEQID NO:1) and short-chain acyl-CoA dehydrogenase form Caenorhabditiselegans (GI 2315796; SEQ ID NO:3), produced using the multisequencealignment program of DNASTAR software (DNASTAR Inc., Madison Wis.).

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to “ahost cell” includes a plurality of such host cells, and a reference to“an antibody” is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are cited for the purpose of describing and disclosing the celllines, vectors, and methodologies which are reported in the publicationsand which might be used in connection with the invention. Nothing hereinis to be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

Definitions

“HSCD,” as used herein, refers to the amino acid sequences ofsubstantially purified HSCD obtained from any species, particularly amammalian species, including bovine, ovine, porcine, murine, equine, andpreferably the human species, from any source, whether natural,synthetic, semi-synthetic, or recombinant.

The term “agonist,” as used herein, refers to a molecule which, whenbound to HSCD, increases or prolongs the duration of the effect of HSCD.Agonists may include proteins, nucleic acids, carbohydrates, or anyother molecules which bind to and modulate the effect of HSCD.

An “allele” or an “allelic sequence,” as these terms are used herein, isan alternative form of the gene encoding HSCD. Alleles may result fromat least one mutation in the nucleic acid sequence and may result inaltered mRNAs or in polypeptides whose structure or function may or maynot be altered. Any given natural or recombinant gene may have none,one, or many allelic forms. Common mutational changes which give rise toalleles are generally ascribed to natural deletions, additions, orsubstitutions of nucleotides. Each of these types of changes may occuralone, or in combination with the others, one or more times in a givensequence.

“Altered” nucleic acid sequences encoding HSCD, as described herein,include those sequences with deletions, insertions, or substitutions ofdifferent nucleotides, resulting in a polynucleotide the same HSCD or apolypeptide with at least one functional characteristic of HSCD.Included within this definition are polymorphisms which may or may notbe readily detectable using a particular oligonucleotide probe of thepolynucleotide encoding HSCD, and improper or unexpected hybridizationto alleles, with a locus other than the normal chromosomal locus for thepolynucleotide sequence encoding HSCD. The encoded protein may also be“altered,” and may contain deletions, insertions, or substitutions ofamino acid residues which produce a silent change and result in afunctionally equivalent HSCD. Deliberate amino acid substitutions may bemade on the basis of similarity in polarity, charge, solubility,hydrophobicity, hydrophilicity, and/or the amphipathic nature of theresidues, as long as the biological or immunological activity of HSCD isretained. For example, negatively charged amino acids may includeaspartic acid and glutamic acid, positively charged amino acids mayinclude lysine and arginine, and amino acids with uncharged polar headgroups having similar hydrophilicity values may include leucine,isoleucine, and valine; glycine and alanine; asparagine and glutamine;serine and threonine; and phenylalanine and tyrosine.

The terms “amino acid” or “amino acid sequence,” as used herein, referto an oligopeptide, peptide, polypeptide, or protein sequence, or afragment of any of these, and to naturally occurring or syntheticmolecules. In this context, “fragments”, “immunogenic fragments”, or“antigenic fragments” refer to fragments of HSCD which are preferablyabout 5 to about 15 amino acids in length and which retain somebiological activity or immunological activity of HSCD. Where “amino acidsequence” is recited herein to refer to an amino acid sequence of anaturally occurring protein molecule, “amino acid sequence” and liketerms are not meant to limit the amino acid sequence to the completenative amino acid sequence associated with the recited protein molecule.

“Amplification,” as used herein, relates to the production of additionalcopies of a nucleic acid sequence. Amplification is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart. (See, e.g., Dieffenbach, C. W. and G. S. Dveksler (1995) PCRPrimer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.,pp.1-5.)

The term “antagonist,” as it is used herein, refers to a molecule which,when bound to HSCD, decreases the amount or the duration of the effectof the biological or immunological activity of HSCD. Antagonists mayinclude proteins, nucleic acids, carbohydrates, antibodies, or any othermolecules which decrease the effect of HSCD.

As used herein, the term “antibody” refers to intact molecules as wellas to fragments thereof, such as Fab, F(ab′)₂, and Fv fragments, whichare capable of binding the epitopic determinant. Antibodies that bindHSCD polypeptides can be prepared using intact polypeptides or usingfragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

The term “antigenic determinant,” as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or a fragment of a protein is usedto immunize a host animal, numerous regions of the protein may inducethe production of antibodies which bind specifically to antigenicdeterminants (given regions or three-dimensional structures on theprotein). An antigenic determinant may compete with the intact antigen(i.e., the immunogen used to elicit the immune response) for binding toan antibody.

The term “antisense,” as used herein, refers to any compositioncontaining a nucleic acid sequence which is complementary to a specificnucleic acid sequence. The term “antisense strand” is used in referenceto a nucleic acid strand that is complementary to the “sense” strand.Antisense molecules may be produced by any method including synthesis ortranscription. Once introduced into a cell, the complementarynucleotides combine with natural sequences produced by the cell to formduplexes and to block either transcription or translation. Thedesignation “negative” can refer to the antisense strand, and thedesignation “positive” can refer to the sense strand.

As used herein, the term “biologically active,” refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, “immunologically active” refers to thecapability of the natural, recombinant, or synthetic HSCD, or of anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The terms “complementary” or “complementarity,” as used herein, refer tothe natural binding of polynucleotides under permissive salt andtemperature conditions by base pairing. For example, the sequence“A-G-T” binds to the complementary sequence “T-C-A.” Complementaritybetween two single-stranded molecules may be “partial,” such that onlysome of the nucleic acids bind, or it may be “complete,” such that totalcomplementarity exists between the single stranded molecules. The degreeof complementarity between nucleic acid strands has significant effectson the efficiency and strength of the hybridization between the nucleicacid strands. This is of particular importance in amplificationreactions, which depend upon binding between nucleic acids strands, andin the design and use of peptide nucleic acid (PNA) molecules.

A “composition comprising a given polynucleotide sequence” or a“composition comprising a given amino acid sequence,” as these terms areused herein, refer broadly to any composition containing the givenpolynucleotide or amino acid sequence. The composition may comprise adry formulation, an aqueous solution, or a sterile composition.Compositions comprising polynucleotide sequences encoding HSCD orfragments of HSCD may be employed as hybridization probes. The probesmay be stored in freeze-dried form and may be associated with astabilizing agent such as a carbohydrate. In hybridizations, the probemay be deployed in an aqueous solution containing salts (e.g., NaCl),detergents (e.g., SDS), and other components (e.g., Denhardt's solution,dry milk, salmon sperm DNA, etc.).

The phrase “consensus sequence,” as used herein, refers to a nucleicacid sequence which has been resequenced to resolve uncalled bases,extended using XL-PCR (Perkin Elmer, Norwalk, Conn.) in the 5′ and/orthe 3′ direction, and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte Clone using a computerprogram for fragment assembly, such as the GELVIEW fragment assemblysystem (GCG, Madison, Wis.). Some sequences have been both extended andassembled to produce the consensus sequence.

As used herein, the term “correlates with expression of apolynucleotide” indicates that the detection of the presence of nucleicacids, the same or related to a nucleic acid sequence encoding HSCD, bynorthern analysis is indicative of the presence of nucleic acidsencoding HSCD in a sample, and thereby correlates with expression of thetranscript from the polynucleotide encoding HSCD.

A “deletion,” as the term is used herein, refers to a change in theamino acid or nucleotide sequence that results in the absence of one ormore amino acid residues or nucleotides.

The term “derivative,” as used herein, refers to the chemicalmodification of HSCD, of a polynucleotide sequence encoding HSCD, or ofa polynucleotide sequence complementary to a polynucleotide sequenceencoding HSCD. Chemical modifications of a polynucleotide sequence caninclude, for example, replacement of hydrogen by an alkyl, acyl, oramino group. A derivative polynucleotide encodes a polypeptide whichretains at least one biological or immunological function of the naturalmolecule. A derivative polypeptide is one modified by glycosylation,pegylation, or any similar process that retains at least one biologicalor immunological function of the polypeptide from which it was derived.

The term “homology,” as used herein, refers to a degree ofcomplementarity. There may be partial homology or complete homology. Theword “identity” may substitute for the word “homology.” A partiallycomplementary sequence that at least partially inhibits an identicalsequence from hybridizing to a target nucleic acid is referred to as“substantially homologous.” The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or northern blot, solutionhybridization, and the like) under conditions of reduced stringency. Asubstantially homologous sequence or hybridization probe will competefor and inhibit the binding of a completely homologous sequence to thetarget sequence under conditions of reduced stringency. This is not tosay that conditions of reduced stringency are such that non-specificbinding is permitted, as reduced stringency conditions require that thebinding of two sequences to one another be a specific (i.e., aselective) interaction. The absence of non-specific binding may betested by the use of a second target sequence which lacks even a partialdegree of complementarity (e.g., less than about 30% homology oridentity). In the absence of non-specific binding, the substantiallyhomologous sequence or probe will not hybridize to the secondnon-complementary target sequence.

The phrases “percent identity” or “% identity” refer to the percentageof sequence similarity found in a comparison of two or more amino acidor nucleic acid sequences. Percent identity can be determinedelectronically, e.g., by using the MEGALIGN program (DNASTAR, Inc.,Madison Wis.). The MEGALIGN program can create alignments between two ormore sequences according to different methods, e.g., the Clustal method.(See, e.g., Higgins, D. G. and P. M. Sharp (1988) Gene 73:237-244.) TheClustal algorithm groups sequences into clusters by examining thedistances between all pairs. The clusters are aligned pairwise and thenin groups. The percentage similarity between two amino acid sequences,e.g., sequence A and sequence B, is calculated by dividing the length ofsequence A, minus the number of gap residues in sequence A, minus thenumber of gap residues in sequence B, into the sum of the residuematches between sequence A and sequence B, times one hundred. Gaps oflow or of no homology between the two amino acid sequences are notincluded in determining percentage similarity. Percent identity betweennucleic acid sequences can also be counted or calculated by othermethods known in the art, e.g., the Jotun Hein method. (See, e.g., Hein,J. (1990) Methods Enzymol. 183:626-645.) Identity between sequences canalso be determined by other methods known in the art, e.g., by varyinghybridization conditions.

“Human artificial chromosomes” (HACs), as described herein, are linearmicrochromosomes which may contain DNA sequences of about 6 kb to 10 Mbin size, and which contain all of the elements required for stablemitotic chromosome segregation and maintenance. (See, e.g., Harrington,J. J. et al. (1997) Nat Genet. 15:345-355.)

The term “humanized antibody,” as used herein, refers to antibodymolecules in which the amino acid sequence in the non-antigen bindingregions has been altered so that the antibody more closely resembles ahuman antibody, and still retains its original binding ability.

“Hybridization,” as the term is used herein, refers to any process bywhich a strand of nucleic acid binds with a complementary strand throughbase pairing.

As used herein, the term “hybridization complex” as used herein, refersto a complex formed between two nucleic acid sequences by virtue of theformation of hydrogen bonds between complementary bases. A hybridizationcomplex may be formed in solution (e.g., C₀t or R₀t analysis) or formedbetween one nucleic acid sequence present in solution and anothernucleic acid sequence immobilized on a solid support (e.g., paper,membranes, filters, chips, pins or glass slides, or any otherappropriate substrate to which cells or their nucleic acids have beenfixed).

The words “insertion” or “addition,” as used herein, refer to changes inan amino acid or nucleotide sequence resulting in the addition of one ormore amino acid residues or nucleotides, respectively, to the sequencefound in the naturally occurring molecule.

“Immune response” can refer to conditions associated with inflammation,trauma, immune disorders, or infectious or genetic disease, etc. Theseconditions can be characterized by expression of various factors, e.g.,cytokines, chemokines, and other signaling molecules, which may affectcellular and systemic defense systems.

The term “microarray,” as used herein, refers to an arrangement ofdistinct polynucleotides arrayed on a substrate, e.g., paper, nylon orany other type of membrane, filter, chip, glass slide, or any othersuitable solid support.

The terms “element” or “array element” as used herein in a microarraycontext, refer to hybridizable polynucleotides arranged on the surfaceof a substrate.

The term “modulate,” as it appears herein, refers to a change in theactivity of HSCD. For example, modulation may cause an increase or adecrease in protein activity, binding characteristics, or any otherbiological, functional, or immunological properties of HSCD.

The phrases “nucleic acid” or “nucleic acid sequence,” as used herein,refer to an oligonucleotide, nucleotide, polynucleotide, or any fragmentthereof, to DNA or RNA of genomic or synthetic origin which may besingle-stranded or double-stranded and may represent the sense or theantisense strand, to peptide nucleic acid (PNA), or to any DNA-like orRNA-like material. In this context, “fragments” refers to those nucleicacid sequences which are greater than about 60 nucleotides in length,and most preferably are at least about 100 nucleotides, at least about1000 nucleotides, or at least about 10,000 nucleotides in length.

The terms “operably associated” or “operably linked,” as used herein,refer to functionally related nucleic acid sequences. A promoter isoperably associated or operably linked with a coding sequence if thepromoter controls the transcription of the encoded polypeptide. Whileoperably associated or operably linked nucleic acid sequences can becontiguous and in reading frame, certain genetic elements, e.g.,repressor genes, are not contiguously linked to the encoded polypeptidebut still bind to operator sequences that control expression of thepolypeptide.

The term “oligonucleotide,” as used herein, refers to a nucleic acidsequence of at least about 6 nucleotides to 60 nucleotides, preferablyabout 15 to 30 nucleotides, and most preferably about 20 to 25nucleotides, which can be used in PCR amplification or in ahybridization assay or microarray. As used herein, the term“oligonucleotide” is substantially equivalent to the terms “amplimer,”“primer,” “oligomer,” and “probe,” as these terms are commonly definedin the art.

“Peptide nucleic acid” (PNA), as used herein, refers to an antisensemolecule or anti-gene agent which comprises an oligonucleotide of atleast about 5 nucleotides in length linked to a peptide backbone ofamino acid residues ending in lysine. The terminal lysine conferssolubility to the composition. PNAs preferentially bind complementarysingle stranded DNA and RNA and stop transcript elongation, and may bepegylated to extend their lifespan in the cell. (See, e.g., Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63.)

The term “sample,” as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acids encoding HSCD,or fragments thereof, or HSCD itself, may comprise a bodily fluid; anextract from a cell, chromosome, organelle, or membrane isolated from acell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a solidsupport; a tissue; a tissue print; etc.

As used herein, the terms “specific binding” or “specifically binding”refer to that interaction between a protein or peptide and an agonist,an antibody, or an antagonist. The interaction is dependent upon thepresence of a particular structure of the protein, e.g., the antigenicdeterminant or epitope, recognized by the binding molecule. For example,if an antibody is specific for epitope “A,” the presence of apolypeptide containing the epitope A, or the presence of free unlabeledA, in a reaction containing free labeled A and the antibody will reducethe amount of labeled A that binds to the antibody.

As used herein, the term “stringent conditions” refers to conditionswhich permit hybridization between polynucleotide sequences and theclaimed polynucleotide sequences. Suitably stringent conditions can bedefined by, for example, the concentrations of salt or formamide in theprehybridization and hybridization solutions, or by the hybridizationtemperature, and are well known in the art. In particular, stringencycan be increased by reducing the concentration of salt, increasing theconcentration of formamide, or raising the hybridization temperature.

For example, hybridization under high stringency conditions could occurin about 50% formamide at about 37° C. to 42° C. Hybridization couldoccur under reduced stringency conditions in about 35% to 25% formamideat about 30° C. to 35° C. In particular, hybridization could occur underhigh stringency conditions at 42° C. in 50% formamide, 5× SSPE, 0.3%SDS, and 200 μg/ml sheared and denatured salmon sperm DNA. Hybridizationcould occur under reduced stringency conditions as described above, butin 35% formamide at a reduced temperature of 35° C. The temperaturerange corresponding to a particular level of stringency can be furthernarrowed by calculating the purine to pyrimidine ratio of the nucleicacid of interest and adjusting the temperature accordingly. Variationson the above ranges and conditions are well known in the art.

The term “substantially purified,” as used herein, refers to nucleicacid or amino acid sequences that are removed from their naturalenvironment and are isolated or separated, and are at least about 60%free, preferably about 75% free, and most preferably about 90% free fromother components with which they are naturally associated.

A “substitution,” as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

“Transformation,” as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. Transformation mayoccur under natural or artificial conditions according to variousmethods well known in the art, and may rely on any known method for theinsertion of foreign nucleic acid sequences into a prokaryotic oreukaryotic host cell. The method for transformation is selected based onthe type of host cell being transformed and may include, but is notlimited to, viral infection, electroporation, heat shock, lipofection,and particle bombardment. The term “transformed” cells includes stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, as well as transiently transformed cells which express theinserted DNA or RNA for limited periods of time.

A “variant” of HSCD, as used herein, refers to an amino acid sequencethat is altered by one or more amino acids. The variant may have“conservative” changes, wherein a substituted amino acid has similarstructural or chemical properties (e.g., replacement of leucine withisoleucine). More rarely, a variant may have “nonconservative” changes(e.g., replacement of glycine with tryptophan). Analogous minorvariations may also include amino acid deletions or insertions, or both.Guidance in determining which amino acid residues may be substituted,inserted, or deleted without abolishing biological or immunologicalactivity may be found using computer programs well known in the art, forexample, DNASTAR software.

The Invention

The invention is based on the discovery of a new human short-chaindehydrogenase (HSCD), the polynucleotides encoding HSCD, and the use ofthese compositions for the diagnosis, treatment, or prevention of immunedisorders and cancer.

Nucleic acids encoding the HSCD of the present invention were firstidentified in Incyte Clone 356351 from the prostate cDNA library(PROSNOT01) using a computer search for amino acid sequence alignments.A consensus sequence, SEQ ID NO:2, was derived from the followingoverlapping and/or extended nucleic acid sequences: Incyte Clones 356351(PROSNOT01), 1852929 (LUNGFET03), 2118849 and 2117677 (BRSTTUT02), and1233166 (LUNGFET03).

In one embodiment, the invention encompasses a polypeptide comprisingthe amino acid sequence of SEQ ID NO:1, as shown in FIGS. 1A, 1B, 1C,and 1D. HSCD is 313 amino acids in length and has four potential caseinkinase II phosphorylation sites at residues S₆₅, T₇₃, S₁₁₄, and S₂₂₄;one potential glycosaminoglycan attachment site at residue S₂₈₆; onepotential microbodies C-terminal targeting signal site at residue S₃₁₁;four potential N-myristoylation sites at residues G₁₄, G₁₈, G₁₆₄, andG₁₉₄; and five potential protein kinase C phosphorylation sites atresidues T₃₇, T₄₃, S₂₃₂, S₂₄₉, and T₃₁₀. As shown in FIG. 2, HSCD haschemical and structural homology with short-chain acyl CoA dehydrogenase(GI 2315796; SEQ ID NO:3). In particular, HSCD and short-chain acyt-CoAdehydrogenase share 43% identity, the N-myristoylation sites at residuesG₁₄ and G₁₈, and the protein kinase C phosphorylation sites at residuesT₃₇ and S₂₄₉. The fragments of SEQ ID NO:2 from about C₂₇₇ to about G286and from about C₂₇₇ to about G₂₈₆ are useful as hybridization probes.Northern analysis shows the expression of this sequence in variouslibraries, at least 50% of which are immortalized or cancerous and atleast 27% of which involve the immune response. Of particular note isthe expression of HSCD in reproductive tissue libraries.

The invention also encompasses HSCD variants. A preferred HSCD variantis one which has at least about 80%, more preferably at least about 90%,and most preferably at least about 95% amino acid sequence identity tothe HSCD amino acid sequence, and which contains at least one functionalor structural characteristic of HSCD.

The invention also encompasses polynucleotides which encode HSCD. In aparticular embodiment, the invention encompasses a polynucleotidesequence comprising the sequence of SEQ ID NO:2, which encodes an HSCD.

The invention also encompasses a variant of a polynucleotide sequenceencoding HSCD. In particular, such a variant polynucleotide sequencewill have at least about 80%, more preferably at least about 90%, andmost preferably at least about 95% polynucleotide sequence identity tothe polynucleotide sequence encoding HSCD. A particular aspect of theinvention encompasses a variant of SEQ ID NO:2 which has at least about80%, more preferably at least about 90%, and most preferably at leastabout 95% polynucleotide sequence identity to SEQ ID NO:2. Any one ofthe polynucleotide variants described above can encode an amino acidsequence which contains at least one functional or structuralcharacteristic of HSCD.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of polynucleotidesequences encoding HSCD, some bearing minimal homology to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention contemplates each and every possiblevariation of polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe polynucleotide sequence of naturally occurring HSCD, and all suchvariations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode HSCD and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring HSCD under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding HSCD or its derivatives possessing a substantially differentcodon usage. Codons may be selected to increase the rate at whichexpression of the peptide occurs in a particular prokaryotic oreukaryotic host in accordance with the frequency with which particularcodons are utilized by the host. Other reasons for substantiallyaltering the nucleotide sequence encoding HSCD and its derivativeswithout altering the encoded amino acid sequences include the productionof RNA transcripts having more desirable properties, such as a greaterhalf-life, than transcripts produced from the naturally occurringsequence.

The invention also encompasses production of DNA sequences which encodeHSCD and HSCD derivatives, or fragments thereof, entirely by syntheticchemistry. After production, the synthetic sequence may be inserted intoany of the many available expression vectors and cell systems usingreagents that are well known in the art. Moreover, synthetic chemistrymay be used to introduce mutations into a sequence encoding HSCD or anyfragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed polynucleotide sequences, and, inparticular, to those shown in SEQ ID NO:2, or a fragment of SEQ ID NO:2,under various conditions of stringency. (See, e.g., Wahl, G. M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; and Kimmel, A. R. (1987)Methods Enzymol. 152:507-511.)

Methods for DNA sequencing are well known and generally available in theart and may be used to practice any of the embodiments of the invention.The methods may employ such enzymes as the Klenow fragment of DNApolymerase I, SEQUENASE® (US Biochemical Corp., Cleveland, Ohio), Taqpolymerase (Perkin Elmer), thermostable T7 polymerase (Amersham,Chicago, Ill.), or combinations of polymerases and proofreadingexonucleases such as those found in the ELONGASE amplification system(GIBCO/BRL, Gaithersburg, Md.). Preferably, the process is automatedwith machines such as the MIGRO LAB 2200 (Hamilton, Reno, Nev.), Peltierthermal cycler (PTC200; MJ Research, Watertown, Mass.), and the ABICatalyst and 373 and 377 DNA Sequencers (Perkin Elmer).

The nucleic acid sequences encoding HSCD may be extended utilizing apartial nucleotide sequence and employing various methods known in theart to detect upstream sequences, such as promoters and regulatoryelements. For example, one method which may be employed,restriction-site PCR, uses universal primers to retrieve unknownsequence adjacent to a known locus. (See, e.g., Sarkar, G. (1993) PCRMethods Applic. 2:318-322.) In particular, genomic DNA is firstamplified in the presence of a primer which is complementary to a linkersequence within the vector and a primer specific to a region of thenucleotide sequenc. The amplified sequences are then subjected to asecond round of PCR with the same linker primer and another specificprimer internal to the first one. Products of each round of PCR aretranscribed with an appropriate RNA polymerase and sequenced usingreverse transcriptase.

Inverse PCR may also be used to amplify or extend sequences usingdivergent primers based on a known region. (See, e.g., Triglia, T. etal. (1988) Nucleic Acids Res. 16:8186.) The primers may be designedusing commercially available software such as OLIGO 4.06 primer analysissoftware (National Biosciences Inc., Plymouth, Minn.) or anotherappropriate program to be about 22 to 30 nucleotides in length, to havea GC content of about 50% or more, and to anneal to the target sequenceat temperatures of about 68° C. to 72° C. The method uses severalrestriction enzymes to generate a suitable fragment in the known regionof a gene. The fragment is then circularized by intramolecular ligationand used as a PCR template.

Another method which may be used is capture PCR, which involves PCRamplification of DNA fragments adjacent to a known sequence in human andyeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.(1991) PCR Methods Applic. 1:111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to place anengineered double-stranded sequence into an unknown fragment of the DNAmolecule before performing PCR. Other methods which may be used toretrieve unknown sequences are known in the art. (See, e.g., Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060.) Additionally, one mayuse PCR, nested primers, and PROMOTERFINDER libraries to walk genomicDNA (Clontech, Palo Alto, Calif.). This process avoids the need toscreen libraries and is useful in finding intron/exon junctions.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. Also,random-primed libraries are preferable in that they will include moresequences which contain the 5′ regions of genes. Use of a randomlyprimed library may be especially preferable for situations in which anoligo d(T) library does not yield a full-length cDNA. Genomic librariesmay be useful for extension of sequence into 5′ non-transcribedregulatory regions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentfluorescent dyes (one for each nucleotide) which are laser activated,and a charge coupled device camera for detection of the emittedwavelengths. Output/light intensity may be converted to electricalsignal using appropriate software (e.g., GENOTYPER and SEQUENCENAVIGATOR, Perkin Elmer), and the entire process from loading of samplesto computer analysis and electronic data display may be computercontrolled. Capillary electrophoresis is especially preferable for thesequencing of small pieces of DNA which might be present in limitedamounts in a particular sample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode HSCD may be used in recombinant DNAmolecules to direct expression of HSCD, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced, and these sequences may be used to clone and expressHSCD.

As will be understood by those of skill in the art, it may beadvantageous to produce HSCD-encoding nucleotide sequences possessingnon-naturally occurring codons. For example, codons preferred by aparticular prokaryotic or eukaryotic host can be selected to increasethe rate of protein expression or to produce an RNA transcript havingdesirable properties, such as a half-life which is longer than that of atranscript generated from the naturally occurring sequence.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alter HSCD-encodingsequences for a variety of reasons including, but not limited to,alterations which modify the cloning, processing, and/or expression ofthe gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example, site-directedmutagenesis may be used to insert new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, introduce mutations, and so forth.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding HSCD may be ligated to aheterologous sequence to encode a fusion protein. For example, to screenpeptide libraries for inhibitors of HSCD activity, it may be useful toencode a chimeric HSCD protein that can be recognized by a commerciallyavailable antibody. A fusion protein may also be engineered to contain acleavage site located between the HSCD encoding sequence and theheterologous protein sequence, so that HSCD may be cleaved and purifiedaway from the heterologous moiety.

In another embodiment, sequences encoding HSCD may be synthesized, inwhole or in part, using chemical methods well known in the art. (See,e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.215-223, and Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser.225-232.) Alternatively, the protein itself may be produced usingchemical methods to synthesize the amino acid sequence of HSCD, or afragment thereof. For example, peptide synthesis can be performed usingvarious solid-phase techniques. (See, e.g., Roberge, J. Y. et al. (1995)Science 269:202-204.) Automated synthesis may be achieved using the ABI431 A peptide synthesizer (Perkin Elmer). Additionally, the amino acidsequence of HSCD, or any part thereof, may be altered during directsynthesis and/or combined with sequences from other proteins, or anypart thereof, to produce a variant polypeptide.

The peptide may be substantially purified by preparative highperformance liquid chromatography. (See, e.g, Chiez, R. M. and F. Z.Regnier (1990) Methods Enzymol. 182:392-421.) The composition of thesynthetic peptides may be confirmed by amino acid analysis or bysequencing. (See, e.g., Creighton, T. (1983) Proteins, Structures andMolecular Properties, W H Freeman and Co., New York, N.Y.)

In order to express a biologically active HSCD, the nucleotide sequencesencoding HSCD or derivatives thereof may be inserted into appropriateexpression vector, i.e., a vector which contains the necessary elementsfor the transcription and translation of the inserted coding sequence.

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding HSCD andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook, J.et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y., ch. 4, 8, and 16-17; and Ausubel, F. M. et al.(1995, and periodic supplements) Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y., ch. 9, 13, and 16.)

A variety of expression vector/host systems may be utilized to containand express sequences encoding HSCD. These include, but are not limitedto, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith virus expression vectors (e.g., baculovirus); plant cell systemstransformed with virus expression vectors (e.g., cauliflower mosaicvirus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

The “control elements” or “regulatory sequences” are thosenon-translated regions, e.g., enhancers, promoters, and 5′ and 3′untranslated regions, of the vector and polynucleotide sequencesencoding HSCD which interact with host cellular proteins to carry outtranscription and translation. Such elements may vary in their strengthand specificity. Depending on the vector system and host utilized, anynumber of suitable transcription and translation elements, includingconstitutive and inducible promoters, may be used. For example, whencloning in bacterial systems, inducible promoters, e.g., hybrid lacZpromoter of the BLUESCRIPT phagemid (Stratagene, La Jolla, Calif.) orPSport1 plasmid (GIBCO/BRL), may be used. The baculovirus polyhedrinpromoter may be used in insect cells. Promoters or enhancers derivedfrom the genomes of plant cells (e.g., heat shock, RUBISCO, and storageprotein genes) or from plant viruses (e.g., viral promoters or leadersequences) may be cloned into the vector. In mammalian cell systems,promoters from mammalian genes or from mammalian viruses are preferable.If it is necessary to generate a cell line that contains multiple copiesof the sequence encoding HSCD, vectors based on SV40 or EBV may be usedwith an appropriate selectable marker.

In bacterial systems, a number of expression vectors may be selecteddepending upon the use intended for HSCD. For example, when largequantities of HSCD are needed for the induction of antibodies, vectorswhich direct high level expression of fusion proteins that are readilypurified may be used. Such vectors include, but are not limited to,multifunctional E. coli cloning and expression vectors such asBLUESCRIPT (Stratagene), in which the sequence encoding HSCD may beligated into the vector in frame with sequences for the amino-terminalMet and the subsequent 7 residues of β-galactosidase so that a hybridprotein is produced, and pIN vectors. (See, e.g., Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509.) pGEX vectors(Pharmacia Biotech, Uppsala, Sweden) may also be used to express foreignpolypeptides as fusion proteins with glutathione S-transferase (GST). Ingeneral, such fusion proteins are soluble and can easily be purifiedfrom lysed cells by adsorption to glutathione-agarose beads followed byelution in the presence of free glutathione. Proteins made in suchsystems may be designed to include heparin, thrombin, or factor XAprotease cleavage sites so that the cloned polypeptide of interest canbe released from the GST moiety at will.

In the yeast Saccharomyces cerevisiae, a number of vectors containingconstitutive or inducible promoters, such as alpha factor, alcoholoxidase, and PGH, may be used. (See, e.g., Ausubel, supra; and Grant etal. (1987) Methods Enzymol. 153:516-544.)

In cases where plant expression vectors are used, the expression ofsequences encoding HSCD may be driven by any of a number of promoters.For example, viral promoters such as the 35S and 19S promoters of CaMVmay be used alone or in combination with the omega leader sequence fromTMV. (Takamatsu, N. (1987) EMBO J. 6:307-311.) Alternatively, plantpromoters such as the small subunit of RUBISCO or heat shock promotersmay be used. (See, e.g., Coruzzi, G. et al. (1984) EMBO J. 3:1671-1680;Broglie, R. et al. (1984) Science 224:838-843; and Winter, J. et al.(1991) Results Probl. Cell Differ. 17:85-105.) These constructs can beintroduced into plant cells by direct DNA transformation orpathogen-mediated transfection. Such techniques are described in anumber of generally available reviews. (See, e.g., Hobbs, S. or Murry,L. E. in McGraw Hill Yearbook of Science and Technology (1992) McGrawHill, New York, N.Y.; pp. 191-196.)

An insect system may also be used to express HSCD. For example, in onesuch system, Autographa californica nuclear polyhedrosis virus (AcNPV)is used as a vector to express foreign genes in Spodoptera frugiperdacells or in Trichoplusia larvae. The sequences encoding HSCD may becloned into a non-essential region of the virus, such as the polyhedringene, and placed under control of the polyhedrin promoter. Successfulinsertion of sequences encoding HSCD will render the polyhedrin geneinactive and produce recombinant virus lacking coat protein. Therecombinant viruses may then be used to infect, for example, S.frugiperda cells or Trichoplusia larvae in which HSCD may be expressed.(See, e.g., Engelhard, E. K. et al. (1994) Proc. Nat. Acad. Sci.91:3224-3227.)

In mammalian host cells, a number of viral-based expression systems maybe utilized. In cases where an adenovirus is used as an expressionvector, sequences encoding HSCD may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain a viable virus which iscapable of expressing HSCD in infected host cells. (See, e.g., Logan, J.and T. Shenk (1984) Proc. Natl. Acad. Sci. 81:3655-3659.) In addition,transcription enhancers, such as the Rous sarcoma virus (RSV) enhancer,may be used to increase expression in mammalian host cells.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained and expressed in aplasmid. HACs of about 6 kb to 10 Mb are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes.

Specific initiation signals may also be used to achieve more efficienttranslation of sequences encoding HSCD. Such signals include the ATGinitiation codon and adjacent sequences. In cases where sequencesencoding HSCD and its initiation codon and upstream sequences areinserted into the appropriate expression vector, no additionaltranscriptional or translational control signals may be needed. However,in cases where only coding sequence, or a fragment thereof, is inserted,exogenous translational control signals including the ATG initiationcodon should be provided. Furthermore, the initiation codon should be inthe correct reading frame to ensure translation of the entire insert.Exogenous translational elements and initiation codons may be of variousorigins, both natural and synthetic. The efficiency of expression may beenhanced by the inclusion of enhancers appropriate for the particularcell system used. (See, e.g., Scharf, D. et al. (1994) Results Probl.Cell Differ. 20:125-162.)

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a “prepro” form of theprotein may also be used to facilitate correct insertion, folding,and/or function. Different host cells which have specific cellularmachinery and characteristic mechanisms for post-translationalactivities (e.g., CHO, HeLa, MDCK, HEK293, and WI38), are available fromthe American Type Culture Collection (ATCC, Bethesda, Md.) and may bechosen to ensure the correct modification and processing of the foreignprotein.

For long term, high yield production of recombinant proteins, stableexpression is preferred. For example, cell lines capable of stablyexpressing HSCD can be transformed using expression vectors which maycontain viral origins of replication and/or endogenous expressionelements and a selectable marker gene on the same or on a separatevector. Following the introduction of the vector, cells may be allowedto grow for about 1 to 2 days in enriched media before being switched toselective media. The purpose of the selectable marker is to conferresistance to selection, and its presence allows growth and recovery ofcells which successfully express the introduced sequences. Resistantclones of stably transformed cells may be proliferated using tissueculture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase genes and adenine phosphoribosyltransferase genes,which can be employed in tk⁻ or apr⁻ cells, respectively. (See, e.g.,Wigler, M. et al. (1977) Cell 11:223-232; and Lowy, I. et al. (1980)Cell 22:817-823) Also, antimetabolite, antibiotic, or herbicideresistance can be used as the basis for selection. For example, dhfrconfers resistance to methotrexate; npt confers resistance to theaminoglycosides neomycin and G-418; and als or pat confer resistance tochlorsulfuron and phosphinotricin acetyltransferase, respectively. (See,e.g., Wigler, M. et al. (1980) Proc. Nati. Acad. Sci. 77:3567-3570;Colbere-Garapin, F. et al (1981) J. Mol. Biol. 150:1-14; and Murry,supra.) Additional selectable genes have been described, e.g., trpB,which allows cells to utilize indole in place of tryptophan, or hisD,which allows cells to utilize histinol in place of histidine. (See,e.g., Hartman, S. C. and R. C. Mulligan (1988) Proc. Natl. Acad. Sci.85:8047-8051.) Visible markers, e.g., anthocyanins, β glucuronidase andits substrate GUS, luciferase and its substrate luciferin may be used.Green fluorescent proteins (GFP) (Clontech, Palo Alto, Calif.) can alsobe used. These markers can be used not only to identify transformants,but also to quantify the amount of transient or stable proteinexpression attributable to a specific vector system. (See, e.g., Rhodes,C. A. et al. (1995) Methods Mol. Biol. 55:121-131.) Although thepresence/absence of marker gene expression suggests that the gene ofinterest is also present, the presence and expression of the gene mayneed to be confirmed. For example, if the sequence encoding HSCD isinserted within a marker gene sequence, transformed cells containingsequences encoding HSCD can be identified by the absence of marker genefunction. Alternatively, a marker gene can be placed in tandem with asequence encoding HSCD under the control of a single promoter.Expression of the marker gene in response to induction or selectionusually indicates expression of the tandem gene as well.

Alternatively, host cells which contain the nucleic acid sequenceencoding HSCD and express HSCD may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations and proteinbioassay or immunoassay techniques which include membrane, solution, orchip based technologies for the detection and/or quantification ofnucleic acid or protein sequences.

The presence of polynucleotide sequences encoding HSCD can be detectedby DNA-DNA or DNA-RNA hybridization or amplification using probes orfragments or fragments of polynucleotides encoding HSCD. Nucleic acidamplification based assays involve the use of oligonucleotides oroligomers based on the sequences encoding HSCD to detect transformantscontaining DNA or RNA encoding HSCD.

A variety of protocols for detecting and measuring the expression ofHSCD, using either polyclonal or monoclonal antibodies specific for theprotein, are known in the art. Examples of such techniques includeenzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs),and fluorescence activated cell sorting (FACS). A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering epitopes on HSCD is preferred, but a competitivebinding assay may be employed. These and other assays are well describedin the art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, aLaboratory Manual, APS Press, St Paul, Minn., Section IV; and Maddox, D.E. et al. (1983) J. Exp. Med. 158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding HSCD includeoligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide. Alternatively, the sequences encoding HSCD,or any fragments thereof, may be cloned into a vector for the productionof an mRNA probe. Such vectors are known in the art, are commerciallyavailable, and may be used to synthesize RNA probes in vitro by additionof an appropriate RNA polymerase such as T7, T3, or SP6 and labelednucleotides. These procedures may be conducted using a variety ofcommercially available kits, such as those provided by Pharmacia &Upjohn (Kalamazoo, Mich.), Promega (Madison, Wis.), and U.S. BiochemicalCorp. (Cleveland, Ohio). Suitable reporter molecules or labels which maybe used for ease of detection include radionuclides, enzymes,fluorescent, chemiluminescent, or chromogenic agents, as well assubstrates, cofactors, inhibitors, magnetic particles, and the like.

Host cells transformed with nucleotide sequences encoding HSCD may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or contained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeHSCD may be designed to contain signal sequences which direct secretionof HSCD through a prokaryotic or eukaryotic cell membrane. Otherconstructions may be used to join sequences encoding HSCD to nucleotidesequences encoding a polypeptide domain which will facilitatepurification of soluble proteins. Such purification facilitating domainsinclude, but are not limited to, metal chelating peptides such ashistidine-tryptophan modules that allow purification on immobilizedmetals, protein A domains that allow purification on immobilizedimmunoglobulin, and the domain utilized in the FLAGS extension/affinitypurification system (Immunex Corp., Seattle, Wash.). The inclusion ofcleavable linker sequences, such as those specific for Factor XA orenterokinase (Invitrogen, San Diego, Calif.), between the purificationdomain and the HSCD encoding sequence may be used to facilitatepurification. One such expression vector provides for expression of afusion protein containing HSCD and a nucleic acid encoding 6 histidineresidues preceding a thioredoxin or an enterokinase cleavage site. Thehistidine residues facilitate purification on immobilized metal ionaffinity chromatography (IMIAC). (See, e.g., Porath, J. et al. (1992)Prot. Exp. Purif. 3: 263-281.) The enterokinase cleavage site provides ameans for purifying HSCD from the fusion protein. (See, e.g., Kroll, D.J. et al. (1993) DNA Cell Biol. 12:441-453.)

Fragments of HSCD may be produced not only by recombinant production,but also by direct peptide synthesis using solid-phase techniques. (See,e.g., Creighton, T. E. (1984) Protein: Structures and MolecularProperties, pp. 55-60, W. H. Freeman and Co., New York, N.Y.) Proteinsynthesis may be performed by manual techniques or by automation.Automated synthesis may be achieved, for example, using the AppliedBiosystems 431A peptide synthesizer (Perkin Elmer). Various fragments ofHSCD may be synthesized separately and then combined to produce the fulllength molecule.

Therapeutics

Chemical and structural homology exists between HSCD and short-chainacyl-CoA dehydrogenase from C. Elegans (GI2315796). In addition, HSCD isexpressed in tissues associated with immune disorders and cancer.Therefore, HSCD appears to play a role in immune disorders and cancer.

Therefore, in one embodiment, an antagonist of HSCD or a fragment orderivative thereof may be administered to a subject to treat or preventa cancer. Such cancers may include, but are not limited to,adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, andteratocarcinoma; and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus. In one aspect, an antibodywhich specifically binds HSCD may be used directly as an antagonist orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissues which express HSCD.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding HSCD may be administered to a subject to treator prevent a cancer including, but not limited to, those describedabove.

Therefore, in another embodiment, an antagonist of HSCD may beadministered to a subject to prevent or treat an immune disorder. Immunedisorders may include, but are not limited to AIDS, Addison's disease,adult respiratory distress syndrome, allergies, anemia, asthma,atherosclerosis, bronchitis, cholecystitis, Crohn's disease, ulcerativecolitis, atopic dermatitis, dermatomyositis, diabetes mellitus,emphysema, erythema nodosum, atrophic gastritis, glomerulonephritis,gout, Graves' disease, hypereosinophilia, irritable bowel syndrome,lupus erythematosus, multiple sclerosis, myasthenia gravis, myocardialor pericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, rheumatoid arthritis, scleroderma, Sjögren's syndrome, andautoimmune thyroiditis; complications of cancer, hemodialysis, andextracorporeal circulation; viral, bacterial, fungal, parasitic,protozoal, and helminthic infections; and trauma. In one aspect, anantibody which specifically binds HSCD may be used directly as anantagonist or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissues which express HSCD.

In another embodiment, a vector expressing the complement of thepolynucleotide encoding HSCD may be administered to a subject to treator prevent an immune disorder including, but not limited to, thosedescribed above.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences, or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of HSCD may be produced using methods which are generallyknown in the art. In particular, purified HSCD may be used to produceantibodies or to screen libraries of pharmaceutical agents to identifythose which specifically bind HSCD. Antibodies to HSCD may also begenerated using methods that are well known in the art. Such antibodiesmay include, but are not limited to, polyclonal, monoclonal, chimeric,and single chain antibodies, Fab fragments, and fragments produced by aFab expression library. Neutralizing antibodies (i.e., those whichinhibit dimer formation) are especially preferred for therapeutic use.

For the production of antibodies, various hosts including goats,rabbits, rats, mice, humans, and others may be immunized by injectionwith HSCD or with any fragment or oligopeptide thereof which hasimmunogenic properties. Depending on the host species, various adjuvantsmay be used to increase immunological response. Such adjuvants include,but are not limited to, Freund's, mineral gels such as aluminumhydroxide, and surface active substances such as lysolecithin, pluronicpolyols, polyanions, peptides, oil emulsions, KLH, and dinitrophenol.Among adjuvants used in humans, BCG (bacilli Calmette-Guerin) andCorynebacterium parvum are especially preferable.

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to HSCD have an amino acid sequence consisting of atleast about 5 amino acids, and, more preferably, of at least about 10amino acids. It is also preferable that these oligopeptides, peptides,or fragments are identical to a portion of the amino acid sequence ofthe natural protein and contain the entire amino acid sequence of asmall, naturally occurring molecule. Short stretches of HSCD amino acidsmay be fused with those of another protein, such as KLH, and antibodiesto the chimeric molecule may be produced.

Monoclonal antibodies to HSCD may be prepared using any technique whichprovides for the production of antibody molecules by continuous celllines in culture. These include, but are not limited to, the hybridomatechnique, the human B-cell hybridoma technique, and the EBV-hybridomatechnique. (See, e.g., Kohler, G. et al. (1975) Nature 256:495-497;Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42; Cote, R. J. etal. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole, S. P. et al.(1984) Mol. Cell Biol. 62:109-120.)

In addition, techniques developed for the production of “chimericantibodies,” such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al.(1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature314:452-454.) Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce HSCD-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries. (See, e.g., Burton D. R. (1991) Proc. Natl. Acad. Sci.88:10134-10137.)

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature.(See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; and Winter, G. et al. (1991) Nature 349:293-299.)

Antibody fragments which contain specific binding sites for HSCD mayalso be generated. For example, such fragments include, but are notlimited to, F(ab′)2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab′)2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (See, e.g., Huse,W. D. et al. (1989) Science 246:1275-1281.)

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity. Numerous protocols for competitivebinding or immunoradiometric assays using either polyclonal ormonoclonal antibodies with established specificities are well known inthe art. Such immunoassays typically involve the measurement of complexformation between HSCD and its specific antibody. A two-site,monoclonal-based immunoassay utilizing monoclonal antibodies reactive totwo non-interfering HSCD epitopes is preferred, but a competitivebinding assay may also be employed. (Maddox, supra.)

In another embodiment of the invention, the polynucleotides encodingHSCD, or any fragment or complement thereof, may be used for therapeuticpurposes. In one aspect, the complement of the polynucleotide encodingHSCD may be used in situations in which it would be desirable to blockthe transcription of the mRNA. In particular, cells may be transformedwith sequences complementary to polynucleotides encoding HSCD. Thus,complementary molecules or fragments may be used to modulate HSCDactivity, or to achieve regulation of gene function. Such technology isnow well known in the art, and sense or antisense oligonucleotides orlarger fragments can be designed from various locations along the codingor control regions of sequences encoding HSCD.

Expression vectors derived from retroviruses, adenoviruses, or herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of nucleotide sequences to the targeted organ, tissue, or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors which will express nucleic acid sequencescomplementary to the polynucleotides of the gene encoding HSCD. (See,e.g., Sambrook, supra; and Ausubel, supra.)

Genes encoding HSCD can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide,or fragment thereof, encoding HSCD. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector, and may last even longer if appropriatereplication elements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5′, or regulatory regions of the gene encodingHSCD. Oligonucleotides derived from the transcription initiation site,e.g., between about positions −10 and +10 from the start site, arepreferred. Similarly, inhibition can be achieved using triple helixbase-pairing methodology. Triple helix pairing is useful because itcauses inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature. (See, e.g., Gee, J. E. et al. (1994)in Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches,Futura Publishing Co., Mt. Kisco, N.Y., pp. 163-177.) A complementarysequence or antisense molecule may also be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodingHSCD.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding HSCD. Such DNAsequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA, constitutivelyor inducibly, can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5′ and/or 3′ ends of the molecule,or the use of phosphorothioate or 2′ O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections, or bypolycationic amino polymers may be achieved using methods which are wellknown in the art. (See, e.g., Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-466.)

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical or sterile composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of HSCD,antibodies to HSCD, and mimetics, agonists, antagonists, or inhibitorsof HSCD. The compositions may be administered alone or in combinationwith at least one other agent, such as a stabilizing compound, which maybe administered in any sterile, biocompatible pharmaceutical carrierincluding, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs, or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with fillers or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks's solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils, such as sesame oil, or synthetic fatty acid esters, such asethyl oleate, triglycerides, or liposomes. Non-lipid polycationic aminopolymers may also be used for delivery. Optionally, the suspension mayalso contain suitable stabilizers or agents to increase the solubilityof the compounds and allow for the preparation of highly concentratedsolutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tendto be more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7%mannitol, at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of HSCD, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells or inanimal models such as mice, rats, rabbits, dogs, or pigs. An animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example HSCD or fragments thereof, antibodies of HSCD,and agonists, antagonists or inhibitors of HSCD, which ameliorates thesymptoms or condition. Therapeutic efficacy and toxicity may bedetermined by standard pharmaceutical procedures in cell cultures orwith experimental animals, such as by calculating the ED50 (the dosetherapeutically effective in 50% of the population) or LD50 (the doselethal to 50% of the population) statistics. The dose ratio oftherapeutic to toxic effects is the therapeutic index, and it can beexpressed as the ED50/LD50 ratio. Pharmaceutical compositions whichexhibit large therapeutic indices are preferred. The data obtained fromcell culture assays and animal studies are used to formulate a range ofdosage for human use. The dosage contained in such compositions ispreferably within a range of circulating concentrations that includesthe ED50 with little or no toxicity. The dosage varies within this rangedepending upon the dosage form employed, the sensitivity of the patient,and the route of administration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting pharmaceuticalcompositions may be administered every 3 to 4 days, every week, orbiweekly depending on the half-life and clearance rate of the particularformulation.

Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to atotal dose of about 1 gram, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically bind HSCD may beused for the diagnosis of disorders characterized by expression of HSCD,or in assays to monitor patients being treated with HSCD or agonists,antagonists, or inhibitors of HSCD. Antibodies useful for diagnosticpurposes may be prepared in the same manner as described above fortherapeutics. Diagnostic assays for HSCD include methods which utilizethe antibody and a label to detect HSCD in human body fluids or inextracts of cells or tissues. The antibodies may be used with or withoutmodification, and may be labeled by covalent or non-covalent attachmentof a reporter molecule. A wide variety of reporter molecules, several ofwhich are described above, are known in the art and may be used.

A variety of protocols for measuring HSCD, including ELISAs, RIAs, andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of HSCD expression. Normal or standard values for HSCDexpression are established by combining body fluids or cell extractstaken from normal mammalian subjects, preferably human, with antibody toHSCD under conditions suitable for complex formation. The amount ofstandard complex formation may be quantitated by various methods,preferably by photometric means. Quantities of HSCD expressed insubject, control, and disease samples from biopsied tissues are comparedwith the standard values. Deviation between standard and subject valuesestablishes the parameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingHSCD may be used for diagnostic purposes. The polynucleotides which maybe used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofHSCD may be correlated with disease. The diagnostic assay may be used todetermine absence, presence, and excess expression of HSCD, and tomonitor regulation of HSCD levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding HSCD or closely related molecules may be used to identifynucleic acid sequences which encode HSCD. The specificity of the probe,whether it is made from a highly specific region, e.g., the 5′regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), will determine whether the probeidentifies only naturally occurring sequences encoding HSCD, alleles, orrelated sequences.

Probes may also be used for the detection of related sequences, andshould preferably have at least 50% sequence identity to any of the HSCDencoding sequences. The hybridization probes of the subject inventionmay be DNA or RNA and may be derived from the sequence of SEQ ID NO:2 orfrom genomic sequences including promoters, enhancers, and introns ofthe HSCD gene.

Means for producing specific hybridization probes for DNAs encoding HSCDinclude the cloning of polynucleotide sequences encoding HSCD or HSCDderivatives into vectors for the production of mRNA probes. Such vectorsare known in the art, are commercially available, and may be used tosynthesize RNA probes in vitro by means of the addition of theappropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³²P or ³⁵S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

Polynucleotide sequences encoding HSCD may be used for the diagnosis ofa disorder associated with expression of HSCD. Examples of such adisorder include, but are not limited to, cancers such asadenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma, andteratocarcinoma; and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus; and immune disorders suchas AIDS, Addison's disease, adult respiratory distress syndrome,allergies, anemia, asthma, atherosclerosis, bronchitis, cholecystitis,Crohn's disease, ulcerative colitis, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, erythema nodosum, atrophic gastritis,glomerulonephritis, gout, Graves' disease, hypereosinophilia, irritablebowel syndrome, lupus erythematosus, multiple sclerosis, myastheniagravis, myocardial or pericardial inflammation, osteoarthritis,osteoporosis, pancreatitis, polymyositis, rheumatoid arthritis,scleroderma, Sjögren's syndrome, and autoimmune thyroiditis;complications of cancer, hemodialysis, and extracorporeal circulation;viral, bacterial, fungal, parasitic, protozoal, and helminthicinfections; and trauma. The polynucleotide sequences encoding HSCD maybe used in Southern or northern analysis, dot blot, or othermembrane-based technologies; in PCR technologies; or in dipstick, pin,ELISA assays or microarrays utilizing fluids or tissues from patientbiopsies to detect altered HSCD expression. Such qualitative orquantitative methods are well known in the art. The polynucleotidesequences encoding HSCD may be used in Southern or northern analysis,dot blot, or other membrane-based technologies; in PCR technologies; indipstick, pin, and ELISA assays; and in microarrays utilizing fluids ortissues from patients to detect altered HSCD expression. Suchqualitative or quantitative methods are well known in the art. Thepolynucleotide sequences encoding HSCD may be used in Southern ornorthern analysis, dot blot, or other membrane-based technologies; inPCR technologies; in dipstick, pin, and ELISA assays; and in microarraysutilizing fluids or tissues from patients to detect altered HSCDexpression. Such qualitative or quantitative methods are well known inthe art.

In a particular aspect, the nucleotide sequences encoding HSCD may beuseful in assays that detect the presence of associated disorders,particularly those mentioned above. The nucleotide sequences encodingHSCD may be labeled by standard methods and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the patient sample is significantlyaltered in comparison to a control sample then the presence of alteredlevels of nucleotide sequences encoding HSCD in the sample indicates thepresence of the associated disorder. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regimen inanimal studies, in clinical trials, or to monitor the treatment of anindividual patient.

In order to provide a basis for the diagnosis of a disorder associatedwith expression of HSCD, a normal or standard profile for expression isestablished. This may be accomplished by combining body fluids or cellextracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, encoding HSCD, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withvalues from an experiment in which a known amount of a substantiallypurified polynucleotide is used. Standard values obtained in this mannermay be compared with values obtained from samples from patients who aresymptomatic for a disorder. Deviation from standard values is used toestablish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis todetermine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding ABBR may involve the use of PCR. These oligomers maybe chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding ABBR, or a fragment of a polynucleotide complementary to thepolynucleotide encoding ABBR, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantitation of closely related DNA or RNA sequences.

Methods which may also be used to quantitate the expression of ABBRinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and interpolating results from standard curves.(See, e.g., Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244;and Duplaa, C. et al. (1993) Anal. Biochem. 212: 229-236.) The speed ofquantitation of multiple samples may be accelerated by running the assayin an ELISA format where the oligomer of interest is presented invarious dilutions and a spectrophotometric or colorimetric responsegives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotide sequences described herein may be used astargets in a microarray. The microarray can be used to monitor theexpression level of large numbers of genes simultaneously and toidentify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, and to develop andmonitor the activities of therapeutic agents.

Microarrays may be prepared, used, and analyzed using methods known inthe art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No.5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci.93:10614-10619; Baldeschweiler et al. (1995) PCT applicationWO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; andHeller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)

In another embodiment of the invention, nucleic acid sequences encodingABBR may be used to generate hybridization probes useful in mapping thenaturally occurring genomic sequence. The sequences may be mapped to aparticular chromosome, to a specific region of a chromosome, or toartificial chromosome constructions, e.g., human artificial chromosomes(HACs), yeast artificial chromosomes (YACs), bacterial artificialchromosomes (BACs), bacterial P1 constructions, or single chromosomecDNA libraries. (See, e.g., Price, C. M. (1993) Blood Rev. 7:127-134;and Trask, B. J. (1991) Trends Genet. 7:149-154.)

Fluorescent in situ hybridization (FISH) may be correlated with otherphysical chromosome mapping techniques and genetic map data. (See, e.g.,Heinz-Ulrich, et al. (1995) in Meyers, R. A. (ed.) Molecular Biology andBiotechnology, VCH Publishers New York, N.Y., pp. 965-968.) Examples ofgenetic map data can be found in various scientific journals or at theOnline Mendelian Inheritance in Man (OMIM) site. Correlation between thelocation of the gene encoding ABBR on a physical chromosomal map and aspecific disorder, or a predisposition to a specific disorder, may helpdefine the region of DNA associated with that disorder. The nucleotidesequences of the invention may be used to detect differences in genesequences among normal, carrier, and affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending genetic maps. Often the placement ofa gene on the chromosome of another mammalian species, such as mouse,may reveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms by physical mapping. This provides valuable informationto investigators searching for disease genes using positional cloning orother gene discovery techniques. Once the disease or syndrome has beencrudely localized by genetic linkage to a particular genomic region,e.g., AT to 11q22-23, any sequences mapping to that area may representassociated or regulatory genes for further investigation. (See, e.g.,Gatti, R. A. et al. (1988) Nature 336:577-580.) The nucleotide sequenceof the subject invention may also be used to detect differences in thechromosomal location due to translocation, inversion, etc., amongnormal, carrier, or affected individuals.

In another embodiment of the invention, ABBR, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes between ABBRand the agent being tested may be measured.

Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest. (See, e.g., Geysen, et al. (1984) PCT applicationWO84/03564.) In this method, large numbers of different small testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. The test compounds are reacted with ABBR, orfragments thereof, and washed. Bound ABBR is then detected by methodswell known in the art. Purified ABBR can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding ABBR specificallycompete with a test compound for binding ABBR. In this manner,antibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with ABBR.

In additional embodiments, the nucleotide sequences which encode ABBRmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I PROSNOT01 cDNA Library Construction

The prostate tissue used for library construction was obtained from a 78year-old Caucasian male with leukemia (Lot No. 94-039, InternationalInstitute for the Advancement of Medicine, Exton Pa.). The tissue wasflash frozen, ground in a mortar and pestle, lysed immediately in buffercontaining guanidinium isothiocyanate and spun through cesium chloride.The lysate was extracted twice with phenol chloroform at pH 8.0 andcentrifuged over a CsCl cushion using an Beckman SW28 rotor in a BeckmanL8-70M Ultracentrifuge (Beckman Instruments). The RNA was precipitatedusing 0.3 M sodium acetate and 2.5 volumes of ethanol, resuspended inwater and DNase treated for 15 min at 37° C. The RNA was isolated usingthe OLIGOTEX kit (QIAGEN Inc., Chatsworth Calif.) and used to constructthe cDNA library.

First strand cDNA synthesis was accomplished using an oligo d(T)primer/linker which also contained an XhoI restriction site. Secondstrand synthesis was performed using a combination of DNA polymerase I,E. coli ligase and RNase H, followed by the addition of an EcoRI adaptorto the blunt ended cDNA. The EcoRI adapted, double-stranded cDNA wasthen digested with XhoI restriction enzyme and fractionated on SEPHACRYLS400 to obtain sequences which exceeded 1000 bp in size. The sizeselected cDNAs were inserted into the LAMBDAZAP vector system(Stratagene, La Jolla Calif.); and the vector, which contains thePBLUESCRIPT phagemid (Stratagene), was transformed into cells of E.coli, strain XL1-BLUEMRF (Stratagene).

The phagemid forms of individual cDNA clones were obtained by the invivo excision process. Enzymes from both PBLUESCRIPT and a cotransformedf1 helper phage nicked the DNA, initiated new DNA synthesis, and createdthe smaller, single-stranded circular phagemid DNA molecules whichcontained the cDNA insert. The phagemid DNA was release, purified, andused to reinfect fresh host cells (SOLR, Stratagene). Presence of thephagemid which carries the gene for β-lactamase allowed transformedbacteria to grow on medium containing ampicillin.

II Isolation and Sequencing of cDNA Clones

Plasmid DNA was released from the cells and purified using the miniprepkit (Catalogue #77468; Advanced Genetic Technologies Corporation,Gaithersburg Md.). This kit consists of a 96 well block with reagentsfor 960 purifications. The recommended protocol was employed except forthe following changes: 1) the 96 wells were each filled with only 1 mlof sterile Terrific Broth (Catalog #22711, GIBCO/BRL, Gaithersburg Md.)with carbenicillin at 25 mg/L and glycerol at 0.4%; 2) the bacteria werecultured for 24 hours after the wells were inoculated and then lysedwith 60 μl of lysis buffer; 3) a centrifugation step employing theBeckman GS-6R @2900 rpm for 5 min was performed before the contents ofthe block were added to the primary filter plate; and 4) the optionalstep of adding isopropanol to TRIS buffer was not routinely performed.After the last step in the protocol, samples were transferred to aBeckman 96-well block for storage.

Alternative methods of purifying plasmid DNA include the use of MAGICMINIPREPS DNA Purification System (Catalogue #A7100, Promega, MadisonWis.), or with QIAWELL 8 plasmid QIAWELL PLUS DNA, and QIAWELL ULTRA DNApurification. Systems (QIAGEN Chatsworth Calif.).

The cDNAs were sequenced by the method of Sanger F and AR Coulson (1975;J Mol Biol 94:441f), using a MICRO LAB 2200 (Hamilton, Reno Nev.) incombination with four Peltier thermal cyclers (PTC200 from MJ Research,Watertown Mass.) and Applied Biosystems 377 or 373 DNA sequencingsystems (Perkin Elmer), and the reading frame was determined.

III Homology Searching of cDNA Clones and Their Deduced Proteins

Each cDNA was compared to sequences in GenBank using a search algorithmdeveloped by Applied Biosystems and incorporated into the INHERIT 670sequence analysis system. In this algorithm, Pattern SpecificationLanguage (TRW Inc, Los Angeles, Calif.) was used to determine regions ofhomology. The three parameters that determine how the sequencecomparisons run were window size, window offset, and error tolerance.Using a combination of these three parameters, the DNA database wassearched for sequences containing regions of homology to the querysequence, and the appropriate sequences were scored with an initialvalue. Subsequently, these homologous regions were examined using dotmatrix homology plots to distinguish regions of homology from chancematches. Smith-Waterman alignments were used to display the results ofthe homology search.

Peptide and protein sequence homologies were ascertained using theINHERIT 670 sequence analysis system using the methods similar to thoseused in DNA sequence homologies. Pattern Specification Language andparameter windows were used to search protein databases for sequencescontaining regions of homology which were scored with an initial value.Dot-matrix homology plots were examined to distinguish regions ofsignificant homology from chance matches.

BLAST, which stands for Basic Local Alignment Search Tool (Altschul, S.F. (1993) J. Mol. Evol. 36:290-300; Altschul et al. (1990) J. Mol. Biol.215:403-410), was used to search for local sequence alignments. BLASTproduces alignments of both nucleotide and amino acid sequences todetermine sequence similarity. Because of the local nature of thealignments, BLAST is especially useful in determining exact matches orin identifying homologs. BLAST is useful for matches which do notcontain gaps. The fundamental unit of BLAST algorithm output is theHigh-scoring Segment Pair (HSP).

An HSP consists of two sequence fragments of arbitrary but equal lengthswhose alignment is locally maximal and for which the alignment scoremeets or exceeds a threshold or cutoff score set by the user. The BLASTapproach is to look for HSPs between a query sequence and a databasesequence, to evaluate the statistical significance of any matches found,and to report only those matches which satisfy the user-selectedthreshold of significance. The parameter E establishes the statisticallysignificant threshold for reporting database sequence matches. E isinterpreted as the upper bound of the expected frequency of chanceoccurrence of an HSP (or set of HSPs) within the context of the entiredatabase search. Any database sequence whose match satisfies E isreported in the program output.

IV. Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; andAusubel, F. M. et al. supra, ch. 4 and 16.)

Analogous computer techniques applying BLAST are used to search foridentical or related molecules in nucleotide databases such as theGenBank or LIFESEQ (Incyte Pharmaceuticals) database. This analysis ismuch faster than multiple membrane-based hybridizations. In addition,the sensitivity of the computer search can be modified to determinewhether any particular match is categorized as exact or homologous.

The basis of the search is the product score, which is defined as:

% sequence identity×% maximum BLAST score 100

The product score takes into account both the degree of similaritybetween two sequences and the length of the sequence match. For example,with a product score of 40, the match will be exact within a 1% to 2%error, and, with a product score of 70, the match will be exact.Homologous molecules are usually identified by selecting those whichshow product scores between 15 and 40, although lower scores mayidentify related molecules.

The results of northern analysis are reported as a list of libraries inwhich the transcript encoding HSCD occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V. Extension of HSCD Encoding Polynucleotides

The nucleic acid sequence of Incyte Clone 356351 was used to designoligonucleotide primers for extending a partial nucleotide sequence tofull length. One primer was synthesized to initiate extension of anantisense polynucleotide, and the other was synthesized to initiateextension of a sense polynucleotide. Primers were used to facilitate theextension of the known sequence “outward” generating ampliconscontaining new unknown nucleotide sequence for the region of interest.The initial primers were designed from the cDNA using OLIGO 4.06software (National Biosciences, Plymouth, Minn.), or another appropriateprogram, to be about 22 to 30 nucleotides in length, to have a GCcontent of about 50% or more, and to anneal to the target sequence attemperatures of about 68° C. to about 72° C. Any stretch of nucleotideswhich would result in hairpin structures and primer-primer dimerizationswas avoided.

Selected human cDNA libraries (GIBCO/BRL) were used to extend thesequence. If more than one extension is necessary or desired, additionalsets of primers are designed to further extend the known region.

High fidelity amplification was obtained by following the instructionsfor the XL-PCR kit (Perkin Elmer) and thoroughly mixing the enzyme andreaction mix. PCR was performed using the Peltier Thermal Cycler(PTC200; M. J. Research, Watertown, Mass.), beginning with 40 pmol ofeach primer and the recommended concentrations of all other componentsof the kit, with the following parameters:

Step 1 94° C. for 1 min (initial denaturation) Step 2 65° C. for 1 minStep 3 68° C. for 6 min Step 4 94° C. for 15 sec Step 5 65° C. for 1 minStep 6 68° C. for 7 min Step 7 Repeat steps 4 through 6 for anadditional 15 cycles Step 8 94° C. for 15 sec Step 9 65° C. for 1 minStep 10 68° C. for 7:15 min Step 11 Repeat steps 8 through 10 for anadditional 12 cycles Step 12 72° C. for 8 min Step 13  4° C. (andholding)

A 5 μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6% to 0.8%) agarosemini-gel to determine which reactions were successful in extending thesequence. Bands thought to contain the largest products were excisedfrom the gel, purified using QIAQUICK (QIAGEN Inc., Chatsworth, Calif.),and trimmed of overhangs using Klenow enzyme to facilitate religationand cloning.

After ethanol precipitation, the products were redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2 to 3 hours, or overnight at 16° C. Competent E. colicells (in 40 μl of appropriate media) were transformed with 3 μl ofligation mixture and cultured in 80 μl of SOC medium. (See, e.g.,Sambrook, supra, Appendix A, p. 2.) After incubation for one hour at 37°C., the E. coli mixture was plated on Luria Bertani (LB) agar (See,e.g., Sambrook, supra, Appendix A, p. 1) containing 2×Carb. Thefollowing day, several colonies were randomly picked from each plate andcultured in 150 μl of liquid LB/2×Carb medium placed in an individualwell of an appropriate commercially-available sterile 96-well microtiterplate. The following day, 5 μl of each overnight culture was transferredinto a non-sterile 96-well plate and, after dilution 1:10 with water, 5μl from each sample was transferred into a PCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction wereadded to each well. Amplification was performed using the followingconditions:

Step 1 94° C. for 60 sec Step 2 94° C. for 20 sec Step 3 55° C. for 30sec Step 4 72° C. for 90 sec Step 5 Repeat steps 2 through 4 for anadditional 29 cycles Step 6 72° C. for 180 sec Step 7  4° C. (andholding)

Aliquots of the PCR reactions were run on agarose gels together withmolecular weight markers. The sizes of the PCR products were compared tothe original partial cDNAs, and appropriate clones were selected,ligated into plasmid, and sequenced.

In like manner, the nucleotide sequence of SEQ ID NO:2 is used to obtain5′ regulatory sequences using the procedure above, oligonucleotidesdesigned for 5′ extension, and an appropriate genomic library.

VI. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:2 are employed to screencDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer, 250 μCi of [γ-³²P] adenosinetriphosphate (Amersham, Chicago, Ill.), and T4 polynucleotide kinase(DuPont NEN, Boston, Mass.). The labeled oligonucleotides aresubstantially purified using a SEPHADEX G-25 superfine resin column(Pharmacia & Upjohn, Kalamazoo, Mich.). An aliquot containing 10⁷ countsper minute of the labeled probe is used in a typical membrane-basedhybridization analysis of human genomic DNA digested with one of thefollowing endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba 1, or Pvu II(DuPont NEN, Boston, Mass.).

The DNA from each digest is fractionated on a 0.7 percent agarose geland transferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR film(Kodak, Rochester, N.Y.) is exposed to the blots or the blots areexposed to film, hybridization patterns are compared visually.

VII. Microarrays

A chemical coupling procedure and an ink jet device can be used tosynthesize array elements on the surface of a substrate. (See, e.g.,Baldeschweiler, supra.) An array analogous to a dot or slot blot mayalso be used to arrange and link elements to the surface of a substrateusing or thermal, UV, mechanical, or chemical bonding procedures, or avacuum system. A typical array may be produced by hand or usingavailable methods and machines and contain any appropriate number ofelements. After hybridization, nonhybridized probes are removed and ascanner used to determine the levels and patterns of fluorescence. Thedegree of complementarity and the relative abundance of each probe whichhybridizes to an element on the microarray may be assessed throughanalysis of the scanned images.

In another alternative, full-length cDNAs or Expressed Sequence Tags(ESTs) comprise the elements of the microarray. Full-length cDNAs orESTs corresponding to one of the nucleotide sequences of the presentinvention, or selected at random from a cDNA library relevent to thepresent invention, are arranged on an appropriate substrate, e.g., aglass slide. The cDNA is fixed to the slide using, e.g., U.V.cross-linking followed, by thermal and chemical and subsequent drying.(See, e.g., Schena, M. et al. (1995) Science 270:467-470; and Shalon, D.et al. (1996) Genome Res. 6:639-645.) Fluorescent probes are preparedand used for hybridization to the elements on the substrate. Thesubstrate is analyzed by procedures described above.

Probe sequences for microarrays may be selected by screening a largenumber of clones from a variety of cDNA libraries in order to findsequences with conserved protein motifs common to genes coding forsignal sequence containing polypeptides. In one embodiment, sequencesidentified from cDNA libraries, are analyzed to identify those genesequences with conserved protein motifs using an appropriate analysisprogram, e.g., the Block 2 Bioanalysis Program (Incyte, Palo Alto,Calif.). This motif analysis program, based on sequence informationcontained in the Swiss-Prot Database and PROSITE, is a method ofdetermining the function of uncharacterized proteins translated fromgenomic or cDNA sequences. (See, e.g., Bairoch, A. et al. (1997) NucleicAcids Res. 25:217-221; and Attwood, T. K. et al. (1997) J. Chem. Inf.Comput. Sci. 37:417-424.) PROSITE may be used to identify functional orstructural domains that cannot be detected using conserved motifs due toextreme sequence divergence. The method is based on weight matrices.Motifs identified by this method are then calibrated against theSWISS-PROT database in order to obtain a measure of the chancedistribution of the matches.

In another embodiment, Hidden Markov models (HMMs) may be used to findshared motifs, specifically consensus sequences. (See, e.g., Pearson, W.R. and D. J. Lipman (1988) Proc. Natl. Acad. Sci. 85:2444-2448; andSmith, T. F. and M. S. Waterman (1981) J. Mol. Biol. 147:195-197.) HMMswere initially developed to examine speech recognition patterns, but arenow being used in a biological context to analyze protein and nucleicacid sequences as well as to model protein structure. (See, e.g., Krogh,A. et al. (1994) J. Mol. Biol. 235:1501-1531; and Collin, M. et al.(1993) Protein Sci. 2:305-314.) HMMs have a formal probabilistic basisand use position-specific scores for amino acids or nucleotides. Thealgorithm continues to incorporate information from newly identifiedsequences to increase its motif analysis capabilities.

VIII. Complementary Polynucleotides

Sequences complementary to the HSCD-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring HSCD. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO 4.06 software andthe coding sequence of HSCD. To inhibit transcription, a complementaryoligonucleotide is designed from the most unique 5′ sequence and used toprevent promoter binding to the coding sequence. To inhibit translation,a complementary oligonucleotide is designed to prevent ribosomal bindingto the HSCD-encoding transcript.

IX. Expression of HSCD

Expression of HSCD is accomplished by subcloning the cDNA into anappropriate vector and transforming the vector into host cells. Thisvector contains an appropriate promoter, e.g., β-galactosidase, upstreamof the cloning site, operably associated with the cDNA of interest.(See, e.g., Sambrook, supra, pp. 404-433; and Rosenberg, M. et al.(1983) Methods Enzymol. 101:123-138.)

Induction of an isolated, transformed bacterial strain with isopropylbeta-D-thiogalactopyranoside (IPTG) using standard methods produces afusion protein which consists of the first 8 residues ofβ-galactosidase, about 5 to 15 residues of linker, and the full lengthprotein. The signal residues direct the secretion of HSCD into bacterialgrowth media which can be used directly in the following assay foractivity.

X. Demonstration of HSCD Activity

The oxidative and reductive CoA dehydrogenase activity is measured with100 μg of protein, 200 pmol of [6,7-³H]17^(beta)-estradiol (or[6,7-³H]estrone for the reduction) acetyl-CoA as a substrate in 100 mMphosphate buffer, pH 7.8 (pH 6.6) with 1uM NAD⁺ as cofactor. Products ofthe reaction are separated on a reversed phase (C 18) high performanceliquid chromatography with mobile phase of acetonitrile:water 1:1 (v/v)as described in (See, e.g., Adamski J. Et. al (1989) Acta Endocr.121:161-167.) Acyl CoA dehydrogenase activity is measured by monitoringNAD⁺ formation at 340 nm using an ultraviolet spectrophotometer.Michaelis-Menten K_(m) values are estimated from initial velocities(conversions of substrate less than 15%) of the corresponding reactions.SDS-PAGE and western blotting is performed as described in (See, e.g.,Adamski, J. Et al. (1992) Biochem. J. 288:375-381.).

XI. Production of HSCD Specific Antibodies

HSCD substantially purified using PAGE electrophoresis (see, e.g.,Harrington, M. G. (1990) Methods Enzymol. 182:488-495), or otherpurification techniques, is used to immunize rabbits and to produceantibodies using standard protocols. The HSCD amino acid sequence isanalyzed using DNASTAR software (DNASTAR Inc.) to determine regions ofhigh immunogenicity, and a corresponding oligopeptide is synthesized andused to raise antibodies by means known to those of skill in the art.Methods for selection of appropriate epitopes, such as those near theC-terminus or in hydrophilic regions are well described in the art.(See, e.g., Ausubel et al. supra, ch. 11.)

Typically, the oligopeptides are 15 residues in length, and aresynthesized using an Applied Biosystems 439A peptide synthesizer usingfmoc-chemistry and coupled to KLH (Sigma, St. Louis, Mo.) by reactionwith N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increaseimmunogenicity. (See, e.g., Ausubel et al. supra.) Rabbits are immunizedwith the oligopeptide-KLH complex in complete Freund's adjuvant.Resulting antisera are tested for antipeptide activity, for example, bybinding the peptide to plastic, blocking with 1% BSA, reacting withrabbit antisera, washing, and reacting with radio-iodinated goatanti-rabbit IgG.

XII. Purification of Naturally Occurring HSCD using Specific Antibodies

Naturally occurring or recombinant HSCD is substantially purified byimmunoaffinity chromatography using antibodies specific for HSCD. Animmunoaffinity column is constructed by covalently coupling anti-HSCDantibody to an activated chromatographic resin, such as CNBr-activatedSEPHAROSE (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Media containing HSCD are passed over the immunoaffinity column, and thecolumn is washed under conditions that allow the preferential absorbanceof HSCD (e.g., high ionic strength buffers in the presence ofdetergent). The column is eluted under conditions that disruptantibody/HSCD binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), and HSCDis collected.

XIII. Identification of Molecules which Interact with HSCD

HSCD, or biologically active fragments thereof, are labeled with ¹²⁵IBolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J.133:529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled HSCD, washed, and anywells with labeled HSCD complex are assayed. Data obtained usingdifferent concentrations of HSCD are used to calculate values for thenumber, affinity, and association of HSCD with the candidate molecules.

Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

3 313 amino acids amino acid single linear PROSNOT01 356351 1 Met AlaAla Pro Met Asn Gly Gln Val Cys Val Val Thr Gly Ala Ser 1 5 10 15 ArgGly Ile Gly Arg Gly Ile Ala Leu Gln Leu Cys Lys Ala Gly Ala 20 25 30 ThrVal Tyr Ile Thr Gly Arg His Leu Asp Thr Leu Arg Val Val Ala 35 40 45 GlnGlu Ala Gln Ser Leu Gly Gly Gln Cys Val Pro Val Val Cys Asp 50 55 60 SerSer Gln Glu Ser Glu Val Arg Thr Leu Phe Glu Gln Val Asp Arg 65 70 75 80Glu Gln Gln Gly Arg Leu Asp Val Leu Val Asn Asn Ala Tyr Ala Gly 85 90 95Val Gln Thr Ile Leu Asn Thr Arg Asn Lys Ala Phe Trp Glu Thr Pro 100 105110 Ala Ser Met Trp Asp Asp Ile Asn Asn Val Gly Leu Arg Gly His Tyr 115120 125 Phe Cys Ser Val Tyr Gly Ala Arg Leu Met Val Pro Ala Gly Gln Gly130 135 140 Leu Ile Val Val Ile Ser Ser Pro Gly Ser Leu Gln Tyr Met PheAsn 145 150 155 160 Val Pro Tyr Gly Val Gly Lys Ala Ala Cys Asp Lys LeuAla Ala Asp 165 170 175 Cys Ala His Glu Leu Arg Arg His Gly Val Ser CysVal Ser Leu Trp 180 185 190 Pro Gly Ile Val Gln Thr Glu Leu Leu Lys GluHis Met Ala Lys Glu 195 200 205 Glu Val Leu Gln Asp Pro Val Leu Lys GlnPhe Lys Ser Ala Phe Ser 210 215 220 Ser Ala Glu Thr Thr Glu Leu Ser GlyLys Cys Val Val Ala Leu Ala 225 230 235 240 Thr Asp Pro Asn Ile Leu SerLeu Ser Gly Lys Val Leu Pro Ser Cys 245 250 255 Asp Leu Ala Arg Arg TyrGly Leu Arg Asp Val Asp Gly Arg Pro Val 260 265 270 Gln Asp Tyr Leu SerLeu Ser Ser Val Leu Ser His Val Ser Gly Leu 275 280 285 Gly Trp Leu AlaSer Tyr Leu Pro Ser Phe Leu Arg Val Pro Lys Trp 290 295 300 Ile Ile AlaLeu Tyr Thr Ser Lys Phe 305 310 1387 base pairs nucleic acid singlelinear PROSNOT01 356351 2 CTAACTTTGG CCTGGGACTC TGCCCCTCTA CCTCAGCACAGAATCGCCCC GGGTCCTACT 60 ACAGAATCAA TCCTTGAACA CTGCCTCCAC GTCGCCGGCTCAATCTGGGC GAGAACCCAG 120 ACTTCCACCG CAGCCCCGCA ATCTGCAGAC CTCAGCGGCAGCGCAGGTGG CAGACCTGCC 180 TCCTTTGCCT GTGAGTCATG GCAGCTCCCA TGAATGGCCAAGTGTGTGTG GTGACTGGTG 240 CCTCCAGGGG TATTGGCCGT GGCATTGCCT TGCAGCTCTGCAAAGCAGGC GCCACAGTTT 300 ACATCACTGG CCGCCATCTG GACACCCTTC GCGTTGTTGCTCAGGAGGCA CAATCCCTCG 360 GGGGCCAATG TGTGCCTGTG GTGTGCGATT CAAGCCAGGAGAGTGAAGTG CGAACGCTGT 420 TTGAGCAAGT GGATCGGGAA CAGCAAGGGC GTCTAGATGTGCTGGTCAAC AATGCTTATG 480 CAGGGGTCCA GACGATCCTG AACACCAGGA ATAAGGCATTCTGGGAAACC CCTGCCTCCA 540 TGTGGGATGA TATCAACAAC GTCGGACTCA GAGGCCACTACTTTTGCTCA GTGTATGGGG 600 CACGGCTGAT GGTACCAGCT GGCCAGGGGC TCATCGTGGTCATCTCCTCC CCAGGAAGCC 660 TGCAGTATAT GTTCAATGTC CCCTATGGTG TGGGCAAAGCTGCGTGTGAC AAGCTGGCTG 720 CTGACTGTGC CCACGAGCTG CGGCGCCATG GGGTCAGCTGTGTGTCTCTG TGGCCGGGGA 780 TTGTGCAGAC AGAACTGCTG AAGGAGCATA TGGCAAAGGAGGAGGTCCTG CAGGATCCTG 840 TGTTGAAGCA GTTCAAATCA GCCTTCTCAT CTGCAGAAACCACAGAATTG AGTGGCAAAT 900 GTGTGGTGGC TTTGGCAACA GATCCCAATA TCCTGAGCCTGAGTGGTAAG GTGCTGCCAT 960 CCTGTGACCT TGCTCGACGC TATGGCCTTC GGGATGTGGACGGCCGCCCC GTCCAAGACT 1020 ATTTGTCTTT GAGCTCTGTT CTCTCACACG TGTCCGGCCTGGGCTGGCTG GCCTCCTACC 1080 TGCCCTCCTT CCTCCGTGTG CCCAAGTGGA TTATTGCCCTCTACACTAGC AAGTTCTAAC 1140 CCTCCTGGTC TGACACTACG TCTCTGCTTG TCTTCTCATTTGGACTTGGT GGTTCGTCCT 1200 GTCTCAGTGA AACAGCAGCC TTTCTTGTTT ACCCATACCCTTGATATGAA GAGAAGCCCT 1260 CTGCTGTGTG TCCGTGGTGA GTTCTGGGGT GCGCCTAGGTCCCTTCTTTG TGCCTTGGTT 1320 TTCCTTGTCC TTCTTTTTAC TTTTTGCCTT AGTATTGAAAAATGCTCTTG GAGCTAATAA 1380 AAGTCTA 1387 323 amino acids amino acidsingle linear GenBank 2315796 3 Met Gly Val Ile Leu Gln Asp Gln Val AlaLeu Val Thr Gly Ala Ser 1 5 10 15 Arg Gly Ile Gly Arg Gly Ile Ala LeuGln Leu Gly Glu Ala Gly Ala 20 25 30 Thr Val Tyr Ile Thr Gly Arg Arg ProGlu Leu Ser Asp Asn Phe Arg 35 40 45 Leu Gly Leu Pro Ser Leu Asp Tyr ValAla Lys Glu Ile Thr Ser Arg 50 55 60 Gly Gly Lys Gly Ile Ala Leu Tyr ValAsp His Ser Asn Met Thr Glu 65 70 75 80 Val Lys Phe Leu Phe Glu Lys IleLys Glu Asp Glu Glu Gly Lys Leu 85 90 95 Asp Ile Leu Val Asn Asn Val TyrAsn Ser Leu Gly Lys Ala Thr Glu 100 105 110 Met Ile Gly Lys Thr Phe PheAsp Gln Asp Pro Ser Phe Trp Asp Asp 115 120 125 Ile Asn Gly Val Gly LeuArg Asn His Tyr Tyr Cys Ser Val Tyr Ala 130 135 140 Ala Arg Met Met ValGlu Arg Arg Lys Gly Leu Ile Val Asn Val Gly 145 150 155 160 Ser Leu GlyGly Leu Lys Tyr Val Phe Asn Val Ala Tyr Gly Ala Gly 165 170 175 Lys GluAla Leu Ala Arg Met Ser Thr Asp Met Ala Val Glu Leu Asn 180 185 190 ProTyr Asn Val Cys Val Val Thr Leu Ile Pro Gly Pro Val Lys Thr 195 200 205Glu Thr Ala Asn Arg Thr Ile Ile Asp Asp Ala Tyr Lys Met Ile Lys 210 215220 Glu Asn Pro Glu Leu Glu Glu Phe Ile Lys Gly Glu Ser Thr Glu Tyr 225230 235 240 Thr Gly Lys Ala Leu Ala Arg Leu Ala Met Asp Pro Gly Lys LeuLys 245 250 255 Lys Ser Gly Lys Thr Leu Phe Thr Glu Asp Leu Ala Gln LysTyr Asp 260 265 270 Phe Ser Asp Lys His Gly Ala Gly Met Glu Pro Gln AsnIle Arg Ser 275 280 285 Ile Arg Thr Ile Leu Gly Thr Met Gly Lys Glu GluVal Ala Lys Tyr 290 295 300 Ile Pro Pro Gln Ile Lys Leu Pro Lys Trp ValIle Trp Gln Ser Val 305 310 315 320 Asn Arg Phe

What is claimed is:
 1. A substantially purified polypeptide comprisingthe amino acid sequence of SEQ ID NO:1.
 2. A composition comprising thepolypeptide of claim 1 and a suitable carrier.
 3. A purified polypeptidecomprising an amino acid sequence selected from the group consisting of:a) an amino acid sequence of SEQ ID NO:1, b) a naturally-occurring aminoacid sequence having at least 90% sequence identity to the sequence ofSEQ ID NO:1, wherein said naturally-occurring amino acid sequence hasCoA dehydrogenase activity, c) a fragment of the amino acid sequence ofSEQ ID NO:1, wherein said fragment has CoA dehydrogenase activity, andd) an immunogenic fragment of 15 contiguous amino acids of the aminoacid sequence of SEQ ID NO:1.
 4. An isolated polypeptide of claim 3,having a sequence of SEQ ID NO:1.
 5. A method for producing apolypeptide of claim 3, the method comprising: a) culturing a cell underconditions suitable for expression of the polypeptide, wherein said cellis transformed with a recombinant polynucleotide, and said recombinantpolynucleotide comprises a promoter sequence operably linked to apolynucleotide encoding the polypeptide of claim 3, and b) recoveringthe polypeptide so expressed.
 6. A method of claim 5, wherein thepolypeptide has the sequence of SEQ ID NO:1.
 7. A composition comprisinga polypeptide of claim 3 and an excipient.
 8. The composition of claim7, wherein the polypeptide has the sequence of SEQ ID NO:1.
 9. A methodfor screening a compound for effectiveness as an agonist of apolypeptide of claim 3, the method comprising: a) exposing a samplecomprising a polypeptide of claim 3 to a compound, and b) detectingagonist activity in the sample.
 10. A method for screening a compoundfor effectiveness as an antagonist of a polypeptide of claim 3, themethod comprising: a) exposing a sample comprising a polypeptide ofclaim 3 to a compound, and b) detecting antagonist activity in thesample.